The task of finding new ways to prevent and treat invasive S. aureus infection, and to develop the requisite understanding of the antibiotic resistance development and spread in this species is urgent. Of the 9 transfers of vancomycin resistance from enterococci to S. aureus, 7 occurred in an outbreak in the Detroit metropolitan area. Subproject 2 is designed to develop an understanding ofthe molecular mechanisms that underiie this outbreak.' Therefore, the following specific aims will be addressed: Specific Aim 1) Determine the relationship between expression of vanA plasmid transfer and replication functions, and transfer rates from E. faecalis or E. faecium to S. aureus. Specific Aim 2) Identify S. aureus plasmid and chromosomal genes that constitute barriers to transfer and establishment of vanA plasmids. Specific Aim 3) Determine how environmental factors, such as biofilm formation or involvement in an infection model, affect vanA transfer. Specific Aim 4) Examine and compare E. faecalis vanA donor, and S. aureus VRSA genome sequences, to identify unanticipated traits that may have contributed to the unusual outbreak of vanA, and for correlation with information derived from the above aims identifying barriers to and promoters of transfer. Relevance to the Program Project Goals and Relation to Other Projects: The overall goal of the program project is to take a well-integrated, multi-disciplinary approach to understand antibiotic resistance development and transmission, and to integrate that effort with the search for novel therapeutics that compromise resistant pathogens, including MRSA. This information will equip us to be alert to and recognize the circumstances that promote the transmission and spread of resistance to this last line antibiotic in S. aureus. RELEVANCE (See instructions): Staphylococcus aureus, especially methicillin resistant (MRSA) strains, have emerged as leading causes of life-threatening infection in the hospital and in the community. While the MRSA problem is alarming, it has become critical as the result ofthe introduction of frank vancomycin resistance. There now have been 9 well documented cases of transfer of vancomycin resistance from enterococci to MRSA in the US.